The present invention is directed to the discovery of compositions that inhibit the nonenzymatic attachment of glucose to albumin, preventing the formation of glycated albumin. The compounds are useful in preventing and treating disorders of the kidney and other organs that result from deleterious effects of glycated albumin.
Glycated albumin, which is formed by the condensation of glucose with reactive protein amino groups, adversely affects capillary function, structure and metabolism. Experimental studies have shown that this glycated protein has distinct biologic effects that the nonglycated counterpart does not possess. These effects include facilitated transport across capillary filtration barriers and hyperfiltration in the kidney, and stimulation of nitric oxide synthase and nitric oxide production, increased synthesis of extracellular matrix proteins, and activation of cytokine and growth factor systems in kidney and vascular tissue. These and other biologic effects of glycated albumin have been described in numerous scientific publications including Kidney International 42:875-881, 1992; Lab Investigation 51:27-35, 1997; Kidney International 45:475-484, 1994; Molecular and Cellular Biochemistry 125:19-25, 1993; Molecular and Cellular Biochemistry 151:61-67, 1995; Kidney International 53:631-638, 1998.
The described biologic activities are observed with concentrations of glycated albumin that found in clinical specimens from human subjects, and do not depend on elevated glucose concentrations to be operative. Since the circulating half-life of albumin in humans is 17 days, there is prolonged exposure of vascular beds to the glycated protein after it is formed.
The use of agents that block the effects of glycated albumin to ameliorate vascular pathologies has been explored in several scientific studies (Kidney International 45:1673-1679, 1994; Journal of Clinical Investigation 95:2338-2345, 1995; Diabetologia 39:270-274, 1996; Journal of Diabetic Complications 12:28-33, 1998). Such agents may be monoclonal antibodies or other molecules that react specifically with fructosyllysine residues that are present on glycated albumin but are not present on nonglycated albumin, and which are disclosed in U.S. Pat. Nos. 5,223,392 and 5,518,720, incorporated by reference herein. Such therapies have been shown, among other things, to prevent the structural and functional changes characteristic of renal and retinal microvascular disease. Anti-glycated albumin therapy therefore has been proposed as a treatment modality for vascular pathologies.
A novel approach to prevent pathologies related to the biologic effects of glycated albumin would be to reduce the formation of the glycated protein and to lower its concentration in the circulation. This could be accomplished by administering compounds that, by binding to specific sites in albumin, can inhibit the attachment of glucose to physiologically important lysine amino groups. The compounds would achieve this desired effect by obscuring the reactive lysine amino group and/or causing a conformational shift in the tertiary structure of the albumin molecule that renders the important glycatable site inaccessible.
Identification of compounds which prevent glycation at physiologically important sites is difficult to accomplish and has not been described in the art. In vitro glycation is distinctly different from in vivo glycation. Excessive concentrations of glucose or reducing carbohydrate are used, and the number of sites that undergo glycation is significantly increased relative to sites which are subject to glycation in vivo. Physiologically important sites are only a small subset of the total number of sites and cannot be distinguished from the unimportant ones after in vitro glycation according to methods described in the art.
Binding to albumin is a likely prerequisite for a compound to prevent glycation of albumin and many compounds of diverse structural classes have been shown to bind to albumin at various sites. Examples include: vitamin C, vitamin E, vitamin B6, diclofenac, acetylsalicylic acid, warfarin, bilirubin, iodobenzoic acids, diazepam, digitoxin, clofibrate, methotrexate, lithium, phenobarbital, cyclosporin benzodiazepine, paracetamol, ibuprofen, suprofen, aminodarone, propanolol, griseofulvan, and others. But binding to albumin is not sufficient for antiglycation activity. Only a few compounds have been reported to influence the condensation of carbohydrate with reactive protein amino groups in vitro, and none of them have been shown to affect lysine amino groups that are physiologically important in vivo or to be of therapeutic benefit when administered in vivo with respect to glycation-related pathologies (Biochemical and Biophysical Research Communications 165:991-996, 1988; Life Sciences 43:1725-1731, 1988; Diabete and Metabolisme 14:40-42, 1988; Biochemica et Biophysica Acta 1120:201-204, 1992; Diabetes 41:167-173, 1992). Moreover, the conditions employed in such in vitro studies are irrelevant to in vivo conditions as to degree of glycation, the reducing sugar and concentration used, and the concentration of compound tested. Binding to albumin and inhibition of in vitro glycation is not synonymous with prevention of glycation at physiologically important sites.
It is a finding of the present invention that many compounds bind to albumin and inhibit glycation at unimportant sites but do not prevent glycation at physiologically important sites.
It is another finding of the present invention that agents that bind to fructosyllysine residues on albumin and, in so doing, prevent pathobiologic effects of glycated albumin provide a useful tool for elucidating which albumin binding ligands are potentially important in preventing the formation of glycated sites that are pathophysiologically important.
The present invention is directed toward discovery of albumin-binding compounds that block non-enzymatic glycation of physiologically important sites which, when glycated, lead to vascular pathologies. The present invention is further directed to methods of use of these novel agents for the treatment of glycation-related pathologies, and novel methods of synthesis of these agents.
The present invention provides novel compounds that inhibit the nonenzymatic glycation of albumin at physiologically relevant sites.
The present invention also provides improved methods of synthesis of 2-phenylamino-phenylacetic acid derivatives.
The present invention further provides novel compositions for preventing and treating glycation-induced pathologies.
These and other objects of the invention are achieved with the discovery of compounds that are reactive with domain(s) in human albumin that are susceptible to nonenzymatic glycation in vivo; that, by binding to the sites in the structure of albumin, protect the protein from attachment to glucose.
The present invention evolved from the finding that glycation-associated pathologies can be ameliorated by ligand compounds that bind to specific glycated sites on albumin. A novel finding of the present invention is that these ligands can be used to identify other compounds that prevent glycation of pathophysiologically important sites in the albumin molecule that are selectively subject to glycation in vivo and that, when glycated, cause deleterious biologic effects in relevant tissues. The present invention further finds that these compounds can be identified by their ability to prevent, in their binding to albumin, the formation of fructosyllysine epitopes in glycated albumin that are recognized by monoclonal antibodies that are site selective for fructosyl-lysine residues that are known to be involved in glycation-associated pathologies.
It is a finding of the present invention that compounds are identified that, by binding to human albumin and protecting the protein from nonenzymatic glycation at pathophysiologically important sites, are therapeutically useful for the treatment of glycation-related pathologies.
Compounds of the present invention are capable of binding to sites in the primary structure of albumin which contain a lysine residue that is a preferential site of nonenzymatic glycation in vivo and/or cause a conformational shift in the tertiary structure of the protein, rendering the glycatable site inaccessible to glucose attachment.
Compounds which are potentially useful are those which are capable of binding albumin and include but are not limited to: vitamin C, vitamin E, vitamin B6, diclofenac, acetylsalicylic acid, warfarin, bilirubin, iodobenzoic acids, diazepam, digitoxin, clofibrate, methotrexate, lithium, phenobarbital, cyclosporin benzodiazepine, paracetamol, ibuprofen, suprofen, aminodarone, propanolol, griseofulvan, and others.
A subset of therapeutically useful compounds can be identified with the monoclonal antibody A717 which binds to fructosyl-lysine residues on albumin and, in so doing, blocks the effects of glycated albumin on vascular pathology. Compounds which prevent the formation of glycated sites recognized by A717 will be therapeutically useful.
Compounds of the present invention possessing this activity are of the structural formula: 
wherein: X is hydrogen, sodium, lithium or potassium and Rxe2x80x23, Rxe2x80x24, Rxe2x80x25, Rxe2x80x26, R2, R3, R4, R5, R6 are the same or different and are hydrogen, chlorine, bromine, fluorine, iodine, methyl, ethyl, propyl, isopropyl, butyl, pentyl, butyloxy, pentyloxy, cyano, thio, methoxy, ethoxy, hydroxy, phosphate, sulfate, nitrate, or amino.
The compounds of the present invention can be tested and selected for low cyclo-oxygenase inhibitory activity and high anti-glycation activity, since some compounds of this structural class have been associated with cyclo-oxygenase inhibitory activity. It is a finding of this invention that this property confers therapeutic advantage by achieving potent inhibition of albumin glycation and lessening untoward side-effects of cyclo-oxygenase inhibition when administered in vivo.
The compounds of the present invention are capable of preventing cellular and tissue damage that is evoked by glycated albumin that is present in the circulation. Since therapeutic concentrations of the compounds of the present invention can inhibit the formation of glycated albumin with high IC50 (the concentration giving 50% inhibition) ratios of anti-glycation to cyclo-oxygenase inhibitory activities, the present invention provides a novel and improved method for the treatment of glycation-related pathologies.
The compounds of this structural class (2-(phenylamino) phenylacetic acids) can be produced following the methods outlined in schemes 1-6. Scheme 1 describes the synthesis of substituted diphenylamines and the subsequent condensation of the appropriate diphenylamines with refluxing chloracetyl chloride to yield the substituted 2-chloro-N-phenylacetanilides. Cyclization is achieved by heating at 160xc2x0 C. in a melt with AlCl3. Hydrolysis of the substituted N-aryloxindoles with NaOH in refluxing ethanolic solution followed by acidification gives the 2-(phenylamino) phenylacetic acids. 
Since alkyl migration and splitting of alkoxy groups can occur during the cyclization reaction above, scheme 2 is beneficial in certain cases. In this scheme substituted diphenylamines are treated with oxalyl chloride in benzene followed by cyclization of the N-phenyloxamic acid chloride with AlCl3 in tetrachloroethane to yield N-arylisatins. Hydrolysis and acidification gives the corresponding phenylglyoxylic acids which are then reduced and acidified to produce the 2-(phenylamino) phenylacetic acids. 
Schemes 1 and 2 are general routes to 2-(phenylamino) phenyl acetic acids, provided that both ortho positions of one phenyl ring of the diphenylamines are occupied in order to avoid the formation of positional isomers of the intermediate oxindoles and isatins. Schemes 3 and 4 are synthetic routes that avoid the formation of isomers. In scheme 3, potassium 2-iodophenyl acetate is reacted with substituted anilines in the presence of potassium carbonate and activated copper powder in hot N-methyl-2-pyrrolidone. Acidification and crystallization yields the 2-(phenylamino) phenylacetic acids. 
Scheme 4 involves the condensation of N,N-dimethyl-2-iodophenylacetamide and anilines in the presence of anhydrous potassium carbonate, copper, and cuprous iodide in refluxing toluene to give the substituted N,N-dimethyl-2-(phenylamino) phenylacetamides. Hydrolysis with KOH in refluxing ethanol followed by acidification yields the 2-(phenylamino) phenylacetic acids. 
Hydroxylated 2-(phenylamio) phenylacetic acids are synthesized from the appropriately substituted methoxy-2-(phenylamino) phenylacetic acids (prepared by scheme 2 and 4). In scheme 5, the methoxy-derivatives are treated with pyridine hydrochloride at 170xc2x0 C. which gives the hydroxy substituted N-phenyloxindoles. Hydrolysis with NaOH in refluxing N-butanol completes the synthesis. 
Hydroxylated compounds with additional methoxy groups are prepared by hydrogenation of he corresponding benzyloxy analogues with Pd-C in tetrahydrofuran and 1,2-dichlorobenzene (scheme 6). 
This invention provides an improved method of synthesis of 2-(phenylamino) phenylacetic acids. Analogous to schemes 3 and 4, scheme 7 produces the 2-(phenylamino) phenylacetic acid from a direct condensation of a phenylacetic acid with an aniline. In this scheme, the phenylacetic acid contains a reactive bromine, the amount of aniline is reduced, the reaction time is reduced, the potassium carbonate is reduced to prevent oxidation of the reaction products, the N-methylpyrrolidone is reduced to accelerate the bimolecular reaction, and reduced amounts of freshly prepared, activated copper is used. This method improved yields of the desired product with little formation of oxyindoles or oxidation products.
This invention also provides therapeutic compositions comprising the above-described compounds.
This invention further provides a method for treating disease comprising administering to the patient an effective amount of a therapeutic composition comprised of the above-described compound(s) capable of inhibiting albumin glycation and a pharmaceutically acceptable carrier therefor.
The present invention also comprises one or more compounds as described above formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection for oral administration in solid or liquid form, for rectal or topical administration, or the like. The compositions can be administered to humans either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracistemally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifuingal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary, pharmaceutically acceptable, excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.
The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
Actual dosage levels of active ingredients in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered to a host in single or divided dose may be in amounts, for example, of from about 1 nanomol to about 100 micromols per kilogram of body weight. Dosage unit compositions may contain such amounts or such submultiples therefor as may be used to make up the daily dose. It will be understood, however, that the specified dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. The dosage level may also depend on patient response as determined by measurement of the concentration of glycated albumin in the circulation at suitable intervals after administration.